ENGLISH ABSTRACT: Phaeomoniella chlamydospora is the main causal organism of Petri disease, which causes
severe decline and dieback of young grapevines (1-7 years old) and also predisposes the wood
for infection by other pathogens. Knowledge about the epidemiology and especially inoculum
sources of this disease is imperative for subsequent development of management strategies.
Through isolation studies it was shown that Pa. chlamydospora is mainly distributed through
infected propagation material in South Africa. However, the infection pathways and inoculum
sources in grapevine nurseries are still unclear. The only existing method to detect this pathogen
in various media is by means of isolation onto artificial growth media. This has proven to be
problematic since this fungus is extremely slow growing (up to 4 weeks from isolation to
identification) and its cultures are often over-grown by co-isolated fungi and bacteria before it
can be identified. The aim of this study was (i) to develop a protocol for the molecular detection
of Pa. chlamydospora in grapevine wood, and (ii) to use this protocol along with others, to test
different samples (water, soil, rootstock and scion cuttings and callusing medium) collected from
nurseries in South Africa at different nursery stages for the presence of Pa. chlamydospora.
A protocol was developed and validated for the molecular detection of Pa.
chlamydospora in grapevine wood. Firstly, several previously published protocols were used to
develop a cost-effective and time-efficient DNA extraction method from rootstock pieces of
potted grapevines. Subsequently, PCR amplification using species-specific primers (Pch1 and
Pch2) was found to be sensitive enough to detect as little as 1 pg of Pa. chlamydospora genomic
DNA from grapevine wood. The protocol was validated using various grapevine material from 3
different rootstock cultivars (101-14 Mgt, Ramsey and Richter 99) collected from each of 3
different nurseries, including grapevines that were subjected to hot water treatment. The basal
end of the rootstock was parallel analysed for Pa. chlamydospora using isolations onto artificial
medium and molecular detection. The identity of PCR products obtained from a subset of
samples, that only tested positive for Pa. chlamydospora based on molecular detection, was
confirmed to be Pa. chlamydospora specific through restriction digestion with AatII. Molecular
detection was found to be considerably more sensitive than isolations, detecting Pa. chlamydospora from samples with positive as well as negative isolations. On average, the
molecular technique detected Pa. chlamydospora in 80.9% of the samples, whereas only 24.1%
of the samples tested positive for Pa. chlamydospora by means of isolations. Pa. chlamydospora
was not isolated from hot water treated samples. The results confirm the importance of hot water
treatment for proactive management of Petri disease in grapevine nurseries. However, Pa.
chlamydospora DNA was molecularly detected in hot water treated samples in frequencies
similar to that detected in non-hot water treated samples. As expected, the DNA in hot water
treated plants was not destroyed and could be detected by the developed molecular detection
protocol. This is an important consideration when using molecular detection for disease
diagnosis or pathogen detection and shows that these methods should be used in conjunction
with other diagnostic tools. Most importantly, the DNA extraction protocol was shown to be 10
to 15 times cheaper than commercial DNA extraction kits.
Preliminary studies showed that the aforementioned molecular detection technique was
not specific and sensitive enough for detection of Pa. chlamydospora in soil and water
(unpublished data). Therefore, a one-tube nested-PCR technique was optimised for detecting Pa.
chlamydospora in DNA extracted from soil, water, callusing medium and grapevine wood.
Rootstock cane sections and soil samples were taking from the mother blocks from several
nurseries. Water samples were collected from hydration and fungicide tanks during pre-storage
and grafting. Scion and rootstock cuttings were also collected during grafting and soil were
collected from the nursery beds prior to planting. The one-tube nested-PCR was sensitive
enough to detect as little as 1 fg of Pa. chlamydospora genomic DNA from water and 10 fg from
wood, callusing medium and soil. PCR analyses of the different nursery samples revealed the
presence of several putative Pa. chlamydospora specific bands (360 bp). Subsequent sequence
analyses and/or restriction enzyme digestions of all 360 bp PCR bands confirmed that all bands
were Pa. chlamydospora specific, except for five bands obtained from callusing media and one
band from water. Considering only Pa. chlamydospora specific PCR bands, the molecular
detection technique revealed the presence of Pa. chlamydospora in 25% of rootstock cane
sections and 17% of the soil samples collected from mother blocks, 42% of rootstock cuttings
collected during grafting, 16% of scion cuttings, 40% of water samples collected after the 12-
hour pre-storage hydration period, 67% of water samples collected during grafting and 8% of the
callusing medium samples. These media should therefore be considered as potential inoculum
sources or infection points of the pathogen during the nursery stages. The results furthermore
confirmed previous findings that Pa. chlamydospora is mainly distributed through infected
rootstock canes and cuttings. Infected scion cuttings were also shown to be potential carriers of the pathogen. Management strategies should include wound protection of rootstock mother
plants, eradicating this pathogen from rootstock-cuttings (e.g. hot water treatment), biological or
chemical amendments in the hydration water and callusing medium and wound protection from
soil borne infections.